Motor neurons and their axons reaching the skeletal muscle have long been considered as the best characterized targets of the degenerative process observed in amyotrophic lateral sclerosis (ALS). However, the involvement of glial cells was also more recently reported. Although oligodendrocytes have been underestimated for a longer time than other cells, they are presently considered as critically involved in axonal injury and also conversely constitute a target for the toxic effects of the degenerative neurons. In the present review, we highlight the recent advances regarding oligodendroglial cell involvement in the pathogenesis of ALS. First, we present the oligodendroglial cells, the process of myelination, and the tight relationship between axons and myelin. The histological abnormalities observed in ALS and animal models of the disease are described, including myelin defects and oligodendroglial accumulation of pathological protein aggregates. Then, we present data that establish the existence of dysfunctional and degenerating oligodendroglial cells, the chain of events resulting in oligodendrocyte degeneration, and the most recent molecular mechanisms supporting oligodendrocyte death and dysfunction. Finally, we review the arguments in support of the primary versus secondary involvement of oligodendrocytes in the disease and discuss the therapeutic perspectives related to oligodendrocyte implication in ALS pathogenesis. Amyotrophic lateral sclerosis (ALS), also known as Charcot’s disease, is a progressive and late-onset neurodegenerative disease that primarily targets motor neurons leading to fatal paralysis . The firWith an incidence of about 2 per 100,000 people per year, the disease comprises 95% of sporadic cases (sALS) and only 5% of familial forms (fALS) . MoleculHowever, pathogenic variants in superoxide dismutase 1 (SOD1), hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9ORF72), fused in sarcoma/translocated in liposarcoma (FUS), and transactive response DNA binding protein 43 (TDBP43) have been the most widely studied . In sALSThe dramatic and fatal evolution of the disease cannot be halted by any therapeutic approach as shown by the inability of most molecules to alleviate the symptoms, with the exception of Riluzole that slightly increases the lifespan of patients ,12,13 anEven though alpha motor neurons and their axons in skeletal muscles are the best-characterized target of the degenerative process, other cells are also affected such as Renshaw cells , corticoIn the present review, we propose to highlight the recent advances regarding the involvement of oligodendroglial cells in the pathogenesis of ALS. We will present the oligodendroglial cells, the process of myelination, and the tight relationship between axons and myelin. Oligodendroglial and white matter abnormalities as well as the chain of deleterious events and molecular mechanisms associated with these abnormalities will be described. Finally, we will discuss the therapeutic perspectives provided by the current data involving the oligodendrocyte in the pathogenesis of ALS.Responsible for axon myelination in the central nervous system (CNS) and indispensable for its normal function, oligodendrocytes are generated in the developing mouse forebrain from oligodendrocyte precursor cells (OPCs) produced in three waves occurring from the embryonic day 12.5 (E12.5) until a last perinatal wave. The last wave arises from Emx1+ progenitors located in the dorsal subependymal area bordering the lateral ventricles and gives rise to the bulk of myelinating oligodendrocytes leading to active myelination of the forebrain during the first three postnatal weeks . In the In the murine brain, early oligodendroglial differentiation is mediated by the down-regulation of Sox2 and the up-regulation of Sox 10, Olig1, and Olig 2. At early stages, human and murine cells also express platelet derived growth factor receptor α (PDGFRα) and NG2 . The transition of OPCs to post-mitotic oligodendrocytes is characterized by chromatin condensation with heterochromatin formation, upregulation of selected microRNAs, and silencing of genes involved in cell proliferation. Loss of PDGFRα/NG2, and increased expression of 2’,3’-Cyclic nucleotide 3’-phosphodiesterase (CNP) and adenomatous polyposis coli (APC), together with Olig 1 and Olig 2, are characteristic of pre-myelinating oligodendrocytes. Finally, myelination triggered by myelin regulatory factor (MYRF) expressed in post-mitotic oligodendrocytes, is accompanied by increased expression of myelin basic protein (MBP), myelin-associated glycoprotein (MAG), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG) ,37. The Although myelinating oligodendrocytes are generated rapidly during the early postnatal period, a small proportion of NG2-expressing OPCs remain thereafter present in both gray and white matter in the adult CNS. OPCs constitute an abundant and widely distributed population of cells that retain the capacity to divide and mature into myelinating cells under physiological and pathological conditions throughout the whole life ,40,41. AMyelination of axons essential for rapid impulse propagation in CNS vertebrates occurs during the late embryonic and early postnatal development successively in the spinal cord and in the brain. White matter is predominantly composed of myelinated and unmyelinated neuronal axons organized into specific tracts with surrounding glial cells. Myelin is composed of lipids (70–85%) and proteins (15–30%) among which the most abundant are MBP and PLP while minor proteins include MOG, CNP, and MAG . In whitElectrical activity derived from the axon is a major regulator of the myelination process. Indeed, activity-dependent release of ATP has been reported to bind the astrocyte purinergic P2 receptor and to induce the release of LIF cytokine, which ultimately stimulates myelination . MoreoveIn the recent years, it has become evident that myelinating cells maintain the long-term integrity and survival of the axons independently of the presence or absence of myelin ,62,63. IAs each oligodendrocyte has the capacity to produce between 20 and 60 myelinating processes and each of the latter displays an internodal length from 20 to 200 µm ,70, the As of 2003, Clement and collaborators revealed that the expression of mutant SOD1 in motor neurons at levels that classically causes disease is not sufficient to trigger all aspects of ALS pathology. Most importantly, non-neuronal cells that do not express mutant SOD1 delay degeneration and extend survival of motor neurons . The useBesides astrocytes and microglia now known to be deeply involved in the progression of ALS ,77, oligG93A transgenic rats revealed ultrastructural disorganization of myelin occurring already in the symptom-free period (60 and 93 days of life) and subsequently worsening in paralyzed animals (120 days of life). These anomalies were associated with decrease of both lipids  and proteins  [G93A mice indicating altered expression of genes involved in different biological processes including apoptosis, oxidative stress, ATP biosynthesis, myelination, and axonal transport [G93A transgenic mice demonstrated a loss of MOG compared with exosomes derived from non-transgenic animals [In the same line, the analysis of myelin in the spinal cord from SOD1LP, MBP) . They weransport . Proteom animals . Demyeli animals .G93A animals revealed decreasing amounts of TDP-43 cells from cervical to thoracic spinal cord segments, mostly detected in the ventral compared to the dorsal horn, exclusively in neurons and oligodendrocytes. The amount of TDP-43 positive cells significantly increased at the onset and progression stages of the disease concurrently with the increase of neuron death in the ventral part of the cervical spinal cord [Besides the detection of TDP-43 cytoplasmic inclusions within oligodendrocytes in the motor cortex and spinal cord of ALS patients ,88,89,90nal cord . A high nal cord .The molecular machinery responsible for cytosolic accumulation of misfolded TDP-43 starts to be uncovered. Indeed, the von Hippel Lindau (VHL)/cullin-2 (CUL2) E3 complex is able to recognize and ubiquitinate misfolded TDP-43 and to promote clearance of the fragmented forms of the protein. However, overexpressed VHL preferentially recognizes misfolded TDP-43 and augments aggregate formation at the juxtanuclear protein quality control center (JUNQ) in agreement with the visualization of cytoplasmic inclusions comprising misfolded TDP-43 and VHL especially in oligodendrocytes in ALS spinal cords. Therefore, the imbalance of VHL/CUL2 may affect oligodendrocytes in ALS by inducing the accumulation of pathogenic proteins, including not only TDP-43, but also mutant SOD1 . FurtherRecently, TDP-43 was found to be required for oligodendroglial physiological functions. Its selective deletion in mature oligodendrocytes in mice revealed the development of progressive neurological symptoms leading to early lethality associated with reduction in myelination. This reduction was proposed to be related to both cell autonomous RIPK1-mediated necroptosis of mature oligodendrocytes and TDP-43–dependent decrease in myelin gene expression. In addition, increased proliferation of OPCs was able to replenish the loss of mature oligodendrocytes, specifically in the white matter. However, degeneration of spinal cord motor neurons was remarkably absent, thus excluding apparent toxicity of oligodendrocyte TDP-43 loss on motor neurons .FUS-containing oligodendroglial cytoplasmic inclusions have been also detected in the CNS of ALS patients exhibiting mutations in the FUS gene. The amount of such inclusions was correlated with disease onset . AnalysiG93A mutant mice analyzed by immunoelectron microscopy [SOD1-containing cytoplasmic inclusions have been also observed in the periaxonal oligodendroglial cytoplasm of SOD1croscopy . Misfoldcroscopy  as well croscopy .Another identified causative gene for autosomal dominant, late-onset fALS, designated as ALS19 and encoding ErBB4, is also at the origin of oligodendroglial inclusions . Like TDG93A mutant mice [After the first descriptions of the existence of aberrant protein aggregates in oligodendrocytes, independent groups successively reported complementary data that accurately described oligodendrocyte dysfunction, progressive gray-matter demyelination, and reactive changes in OPCs observed in the motor cortex and spinal cord of both sALS and fALS patients as well as in SOD1ant mice ,89,107 , which displays an increased level in the spinal cord from ALS patients and SOD1 disease . Anotherstigated .Future works should delineate whether a pro-inflammatory OPC phenotype is present in ALS tissues. This phenotype has been shown to occur in response to inflammatory cues, to promote tissue damage and to block remyelination ,114. ThuG93A mice, suggesting that viable and uninjured axons were remyelinated. Ultrastructural anomalies typical of Wallerian degeneration were also observed together with MBP immunoreactivity more diffuse in the end stage mutant than in the control. Axons with mature myelin displayed a thicker sheath than controls possibly reflecting axonal shrinkage. Myelin debris and immature myelin sheaths could also be detected [The clear reduction of myelination in ALS corticospinal tracts and ventral horn gray matter of the spinal cord was obviously consistent with the finding that the newly generated oligodendrocytes were dysfunctional and failed both in terms of myelin synthesis and trophic support as suggested by the lower expression of MBP and MCT1, respectively ,107. EleG37R from OPCs and their progeny delayed disease onset and prolonged survival, confirming the direct relationship with motor neuron degeneration. SOD1 removal helped to preserve MCT1 expression in some mice at early stages of the disease indicating that expression of mutant SOD1 in NG2+ cells and their oligodendrocyte progeny has a deleterious effect on motor neuron survival. Thus, the progressive loss of gray matter oligodendrocytes as well as the failure to restore these critical cells were proposed to accelerate disease progression in ALS by depriving motor neurons of essential metabolic support [Importantly, the genetic deletion of mutant human SOD1 support .G93A oligodendrocytes or human ALS oligodendrocytes derived from multiple genetic and sporadic forms of the disease induce motor neuron death via soluble and insoluble factors that require cell-to-cell contact or very close proximity. Interestingly, the toxic effects can be rescued by reducing SOD1 in OPCs (but not in differentiated oligodendrocytes) except for OPCs derived from cells carrying the C9ORF72 mutations, which seem to be SOD1 independent and thus define a discrete subgroup of ALS patients [In vitro models of motor neuron and oligodendrocyte cocultures led to consistent results indicating that mouse SOD1patients . In agrepatients . Howeverpatients .Still in regard to disease heterogeneity, a yet higher level of complexity was recently provided by transcriptome arrays performed in anterior horn of the spinal cord and frontal cortex from sALS cases that revealed striking regional differences. In the spinal cord, up- and down-regulated clusters were related to inflammation/apoptosis and axoneme structures/protein synthesis, respectively. In the frontal cortex, up-regulated gene clusters involved neurotransmission, synaptic proteins, and vesicle trafficking, whereas down-regulated genes clustered into oligodendrocyte function and myelin-related proteins even though patients were at stages with no apparent impairment .Since motor neuron degeneration in both spinal cord and motor cortex is an essential and furthermore necessary component of the disease to confirm diagnosis of ALS, glutamate excitotoxicity was among the first mechanisms implicated in oligodendrocyte pathogenesis in ALS. Indeed, when motor neurons start to degenerate, they release high levels of glutamate . OligodeOxidative stress induced by an imbalance of free radicals is also a major contributor to ALS neurodegeneration. It exacerbates homeostatic dysregulation of mitochondria, proteins, genes, and other cellular processes, which are the subject of continuous investigations. The subsequent alteration of protein folding is consistent with the formation of protein aggregates promoted by mutations in ALS-related genes including SOD1, FUS, TDP-43, and ErBB4 as described above. These aggregates cause further endoplasmic reticulum (ER) stress and cell apoptosis as namely suggested for the SOD1 mutant . In agreTo resolve ER stress and defect in protein homeostasis, cells trigger intrinsic mechanisms called unfolded protein response (UPR) and heat shock response (HSR), two of the major signaling pathways implicated in the restoration of protein homeostasis. In ALS human post-mortem tissues, UPR is activated in both the motor cortex and spinal cord with specific expression of select UPR target genes, such as PDIs (protein disulfide isomerase) observed in motor cortex of sALS cases where these genes strongly correlate with oligodendrocyte markers. In contrast, the ER-associated degradation (ERAD) and HSR genes mostly activated in spinal cord, correlate with the expression of neuronal markers evidencing cell-type-specific contributions. Interestingly, an increase in PDI expression has been reported to occur during active myelination in cortical regions of rats. In line with this observation, a strong PDI expression in oligodendrocytes was observed in SOD1 mice in agreement with abnormalities in plasma and membrane lipid signaling, especially in the early symptomatic stages of ALS . DespiteIn ALS models, oxidative damage to mRNA has been reported in oligodendrocytes before the symptomatic stage when microgliosis is not yet detected. Among the most vulnerable mRNAs are those involved in mitochondrial electron transport and myelination. Thus, oxidative damage to MBP mRNA likely accounts for reduced MBP expression D consistA growing literature is currently providing evidence for the involvement of extrinsic mechanisms originating in neighboring glial cells and related to the existence of a dual activity for both reactive astrocytes and microglia in inflammatory diseases of the CNS, namely demyelinating diseases (for review ). ReactiAll these modifications were particularly evident in the dysmorphic oligodendrocytes accumulating at the anterior horns suggesting a causative relationship between mutant SOD1 and connexin pathology. They were also consistent with the stage-dependent progression of astrogliosis and microglial activation in the anterior horns of the same model. In normal conditions, oligodendrocytes can import glucose through GLUT1 and connexin junctions for glycolysis. The latter can yield sufficient ATP to support oligodendrocyte survival while the aerobic glycolysis product, lactate, can be transferred to axons via MCT1 and used as an energy source for axonal activity. Therefore, loss of membranous Cx47 and Cx32 in oligodendrocytes in mSOD1 ALS model mice may lead to insufficient glucose supply both contributing to oligodendrocyte damage and motor neuron death through energy failure . These fLike astrocytes, microglia may be endowed with detrimental effects on oligodendrocytes and myelin via the synthesis of cytokines, chemokines, cell adhesion glycoproteins, or reactive oxygen radicals able to damage axons, myelin, oligodendrocytes, and thus implicated in the initiation and propagation of the inflammatory cascade contributing to demyelination. Several recent publications have addressed the topic ,134. HowThe observation that oligodendrocyte degeneration occurs earlier than motor neuron loss ,107 has A recent work also supported the idea that oligodendrocyte and myelin pathology outstrip neuronal degeneration in some regions by analyzing the extent of inclusion pathology in post-mortem tissues from human sALS and mutant C9ORF72-related ALS (C9ALS) cases . Data reOther arguments supporting the primary involvement of oligodendrocytes were provided by the selective deletion of mutant SOD1 protein from the oligodendroglial cells that was found to delay disease onset and improve mouse survival, probably suggesting a key role for oligodendroglial cells in accelerating injury to vulnerable motor neurons. However, the time between symptom onset and death remained unmodified, suggesting that the longer survival time was only related to the delay in disease onset. Furthermore, the role of oligodendrocytes in supporting axonal energy metabolism is also obviously consistent with a major role of oligodendrocytes in the deterioration of motor neurons. In a consistent manner, SOD1 removal from oligodendrocytes led to MCT1 levels comparable to those found in the wild-type mice as well as to a lower level of reactive astrocytes and microglia that may also possibly participate in the delay in disease onset since alterations in astrocytes and microglia are critical for determining disease onset ,24. In aOn the contrary, a few other works question the primary involvement of oligodendrocytes in the pathological propagation of the disease.First, the neuronal expression of mutant SOD1 was found to cause motor neuron degeneration and paralysis in transgenic mice displaying cytosolic dendritic ubiquitinated SOD1 aggregates as a dominant pathological feature. Moreover, the crossing of neuron-specific mutant SOD1 mice with ubiquitously wild-type SOD1-expressing mice led to high wild-type SOD1 aggregation in oligodendrocytes after the onset of neuronal degeneration. The chronologic steps were thus that mutant SOD1 in neurons triggers neuronal degeneration, which subsequently may facilitate aggregate formation in surrounding glial cells .Second, the spread of the pathology into four stages has been initially proposed to occur through corticofugal axonal transmission consistent with the detection of oligodendroglial TDP-43 aggregates along with neuronal inclusions. However, while TDP-43 immunoreactive oligodendrocytes were frequently observed in the white matter under the motor and sensory cortices, no TDP-43 pathology was detected, namely in the white matter along the corticospinal tract of the same tissue samples, .G37R. Motor neuron damages included synapse and metabolic abnormalities as well as endoplasmic reticulum stress. Glia-related changes occurred early in astrocytes in genes involved in inflammation/metabolism or standing as targets of the lipid-activated transcription factors PPAR and LXR. Dysregulation of myelination and lipid signaling pathways together with activation of ETS transcription factors appeared in oligodendrocytes only after disease initiation. Gene expression changes were most dramatic in motor neurons (260 significant changes), compared to astrocytes (108) and oligodendrocytes (23 including 14 up-regulated and 9 down-regulated RNAs). Notably, ER chaperones PDI and FKBP9 mRNA levels were much more highly up-regulated in astrocytes and oligodendrocytes compared with motor neurons, suggesting that motor neurons could be intrinsically more vulnerable to unfolded protein accumulation. Unexpectedly, MCT1 decrease previously shown in SOD1G93A mice [Third, high-throughput RNA sequencing together with translating ribosome affinity purification (TRAP) provided evidence that pathogenesis involves a temporal cascade of cell type-selective damage starting in motor neurons, with subsequent damage within glia that might subsequently drive disease propagation. The data were obtained in mice expressing a moderate level of the fALS-causing mutation SOD193A mice  was not 93A mice .Fourth, the transfer of fluorescent wild-type or mutant SOD1 cytosolic proteins occurring first in motor neurons and then in neighboring oligodendrocytes in the gray matter of the ventral spinal cord is still in support of the primary involvement of motor neurons and not oligodendrocytes . The demIt is quite clear that our current knowledge does not allow to conclude on the primary or secondary involvement of oligodendrocytes or motor neurons in ALS. However, a few arguments quite inconsistent with the hypothesis that neurons are primarily involved can be pointed out. Indeed, the increase in oligodendrogenesis reported in the ALS models does not support this hypothesis. First, the production of new oligodendrocytes without target axons to myelinate would be inadequate. Second, as motor axons comprise only a minor proportion of myelinated fibers in the spinal cord and as a single oligodendrocyte can synthesise numerous internodal segments on different axons, healthy non-motor axons should allow oligodendrocyte survival. Third, the observation of ultrastructurally normal axons with immature myelin sheaths in the ventral gray matter of symptomatic SOD1 mutant mice is in favor of the attempt of oligodendrocytes to remyelinate viable axons, rather than to degenerate due to axon loss . For allIn the spinal cord, an additional hypothesis might be the involvement of an aberrant regulation of Olig2 function, the transcription factor expressed by the progenitor cells that give rise successively to both the motor neurons and the oligodendrocytes in the pMN domain during the embryonic development. In support of this hypothesis, the characterization of ChIP-Seq-based Olig2 target genes in this domain was found to be relevant to the pathogenesis of ALS. Indeed, 740 Olig2 target genes overlapping between the newly generated motor neurons and OPCs have been characterized to be closely related to “alternative splicing” in agreement with increasing evidence indicating that RNA metabolism, including the regulation of transcription and alternative splicing, is profoundly disturbed in ALS. Molecular network analysis also suggested that Olig2 down-regulates a wide range of target genes involved in diverse neuronal and glial functions . FurtherG93A mice. In addition, in the ShhOlig2−/− mice, the morphologically abnormal OPCs failed to participate in the synaptic remodeling of motor neurons in response to motor neuron injury. Therefore, the common characteristic between OPCs in SOD1G93A and ShhOlig2−/− animals is that OPCs are forced to over-proliferate to compensate for either OPC degeneration or reduced ventral OPC production, respectively. Thus, a proposed hypothesis is that OPCs in the ALS models might become progressively unable to contribute to synaptic remodeling of distressed motor neurons [Still in the context of embryonic development, the increase in OPC proliferation observed in ALS was found to remind the phenotype observed upon the partial ablation of Sonic Hedgehog (Shh) signaling in the Olig2-expressing progenitors. Shh signaling pathway is well characterized for its contribution to the generation of progenitor cells, which give rise to both motor neurons and OPCs during the embryonic development of the spinal cord. In ShhOlig2−/− transgenic mice, the decrease of OPC production in the ventral spinal cord led dorsal OPCs to populate the entire spinal cord and to take on an altered morphology proposed to be similar to the one found in the vicinity of motor neurons in the SOD1 neurons . HoweverG93A. The molecule is currently in Phase III trial [Over the past 20 years, clinical trials in ALS patients have reached limited success likely due to the complex nature of this disease. Various compounds with different mechanisms of action have been studied including Riluzole and Edaravone that constitute the only current treatments for ALS, nevertheless with mild benefits if one considers survival and quality of life . RiluzolII trial ,149.Moreover, the most recent therapeutic perspective came from antisense oligonucleotides, which are the first approved drugs to treat spinal muscular atrophy and are currently proposed to be potentially interesting in ALS treatment ,151. TheNone of the molecules mentioned above specifically targets the oligodendrocytes. However, several therapeutic avenues are currently investigated in this purpose. They can be divided into a series of approaches requiring further technical or conceptual improvements on one side and the use of promyelinating molecules, which likely stand as the most promising approach in not too a distant future, on the other side. The most important out of these approaches are listed in ®) into the lumbar/cervical spinal cord of ALS patients has been proposed. These cells represent one of the earliest precursors within the oligodendroglial and astroglial cell lineage and their usage in ALS therapy might be a beneficial approach resulting in the enrichment of nervous system by healthy donor-derived cells [The dysfunction of oligodendrocytes and the cytoplasmic deposits of ALS proteins in these cells (that may be or not one of the causes of the disease) led to propose cell transplantation in order to replenish the lost and/or defective oligodendrocytes. Recently, the transplantation of human glial restricted progenitor cells  in the spinal cord, likely responsible for mitigating disease progression and increasing animal survival [Finally, although neither directly linked to oligodendrocyte involvement in ALS pathogenesis nor usable as a possible therapeutic perspective, an amazing observation has reported that myelin antigen-mediated immunization of the SOD1survival .Since oligodendrocyte degeneration and death stand as major features of ALS impairing both axonal conductance and metabolic support to neurons, molecules endowed with remyelinating properties might thus restore both defects. The development of such molecules constitutes the current challenge in the therapeutic approach of multiple sclerosis, the most common demyelinating disease of the CNS. In this regard, several targets have been identified during the last years for promoting myelin regeneration in the brain and spinal cord. They include modulators of sphingosine-1-phosphate receptor, muscarinic M1 receptor, histamine H1 receptors, retinoid X receptor γ, or opioid κ-receptors; inhibitors of γ-secretase blocking Notch, leukotriene, or Lingo pathways; modulators of hormone signaling such as androgens, estrogens, tri-iodothyronine, and thyroxine; antipsychotics (quetiapine); antifungals (miconazole); and steroids (clobetasol). Only a few have reached clinical trials of myelin regeneration, which have mostly recruited patients suffering from optic neuritis, a common symptom of multiple sclerosis, often occurring during the early stage of the pathology. The first results indicate some promising outcomes in these patients .G93A mice and was found to reduce microgliosis, modulate microglia-related inflammatory genes, and enhance motor neuron survival [Whether outcomes in the optic nerve can be extrapolated to other CNS areas remains unknown. However, targets that are currently considered for multiple sclerosis may be obviously worth exploring in ALS. A few molecules are currently evaluated in the latter . For inssurvival  further G93A mice compared to healthy animals. This increase started at the pre-symptomatic stage and was exacerbated at the late symptomatic phase suggesting an altered timing of GPR17 expression. Indeed, GPR17 expression is known to be the result of the complex integration of intrinsic determinants regulating oligodendroglial differentiation with extracellular stimuli acting on the Gpr17 gene. Thus, the maintenance of a high GPR17 expression was proposed to impede proper differentiation of OPCs. Interestingly, the defective differentiation of OPCs isolated from the spinal cord of the SOD1G93A mutant mice was rescued by treatment with the GPR17 antagonist montelukast (a leukotrien receptor antagonist authorized for the management of asthma). This already-marketed, orally available drug may represent a multi-target drug with high translational potential for repurposing strategies namely for ALS management [Moreover, the P2Y—like G protein-coupled receptor 17 (GPR17), which has been previously characterized as a good pharmacological target to implement repair and remyelination under several neurodegenerative conditions including multiple sclerosis, was reported to be expressed at a much higher level in the spinal cord from SOD1nagement . This wonagement . Again, nagement . ObviousAlthough remyelinating molecules seem able to reach pre-clinical and clinical development before the other approaches listed above, they will preferentially have to be endowed with multi-target activities if we consider for instance the involvement of microglia and astrocytes in the inflammatory process contributing to oligodendrocyte degeneration ,130,174.The critical role of oligodendrocytes in ALS is presently indisputable. The degeneration of oligodendrocytes during disease progression together with the increased proliferation of OPCs without full differentiation into mature oligodendrocytes obviously participate in the whole spectrum of damages contributing to neurodegeneration. The failure of metabolic support provided to axons and the high level of demyelination directly related to the defective oligodendroglial lineage should both precipitate the degeneration of axons and death of neurons. Conversely, motor neuron degeneration may also contribute to make surrounding oligodendrocytes more vulnerable due to the high energy requirement and damaging oxidative reactions. The key players of the signaling pathways up to now identified as being implicated in oligodendroglial cell alterations during ALS disease already provide a valuable source of molecular targets to develop new cell type selective therapeutic approaches. In addition, since our current knowledge regarding both glial heterogeneity and cellular communication is conspicuously facilitated by highly performant ’OMIC’ approaches as shown in several other neurodegenerative diseases, one may consider that specific transcriptomic changes in cells displaying toxic ALS-related protein aggregates and in closely neighboring cells should undoubtedly improve our understanding of the oligodendrocyte communication with other glial or neuronal cells as well as their impact on disease progression. Similarly, the emergence of human iPSC models for ALS together with gene editing technology will allow investigation of the intricate crosstalk between the various cell types including oligodendrocytes in the human disease.


Living species are continuously subjected to all extrinsic forms of reactive oxidants and others that are produced endogenously. There is extensive literature on the generation and effects of reactive oxygen species (ROS) in biological processes, both in terms of alteration and their role in cellular signaling and regulatory pathways. Cells produce ROS as a controlled physiological process, but increasing ROS becomes pathological and leads to oxidative stress and disease. The induction of oxidative stress is an imbalance between the production of radical species and the antioxidant defense systems, which can cause damage to cellular biomolecules, including lipids, proteins and DNA. Cellular and biochemical experiments have been complemented in various ways to explain the biological chemistry of ROS oxidants. However, it is often unclear how this translates into chemical reactions involving redox changes. This review addresses this question and includes a robust mechanistic explanation of the chemical reactions of ROS and oxidative stress. In chemistry, a free radical (FR) is a relatively stable species that contains one or more unpaired electrons and can react with other molecules, either by donating its unpaired electron to another molecule or by taking it away from another molecule to increase stability. In this way it converts the molecule with which it reacts into another FR, so a common feature of FR reactions is the chain process: one radical gives rise to another radical. This process only ceases when two FRs react with each other.2 molecule to accept electrons [•O2−), hydrogen peroxide (H2O2), hydroxyl radical (OH−), and singlet oxygen (1O2−), produced by all kinds of cells. Stationary  levels of ROS are intrinsic to the normal functioning of cells, fulfilling the functions of cell signalling and homeostasis [In a biological context, reactive oxygen species (ROS) are formed as a natural by-product of cellular aerobic metabolism. Mitochondrial respiration is a significant cause of reactive oxygen species (ROS) . In addilectrons , generateostasis . On the eostasis .2 for efficient energy production. Molecular O2 contains two unpaired electrons, but it is weakly reactive because they are located in different molecular orbitals and have a parallel spin. Consequently, oxygen preferentially accepts electrons one at a time. The ultimate goal of the electron transport chain ETC in the inner mitochondrial membrane (complexes I to V) is the reduction of the oxygen molecule to produce water. The first step of the O2 reduction reaction occurs spontaneously  [•O2− in large quantities for use in the oxygen-dependent destruction mechanisms of invading pathogens [Superoxide ain ETC) , where aathogens .•OH (−9 s) and high reactivity.The hydroxyl radical •OH A–C, the 2 and ROS reduction are reactions in which the final product is water and have a negative Gibbs free energy, ∆Go ≤ 0, so they occur spontaneously [2 to superoxide ion •O2−, the precursor of most reactive oxygen species.Oaneously . A smallROS can be induced by exogenous sources such as tobacco, pollution, smoke, drugs, xenobiotics or ionising radiation, leading to irreversible effects on tissue development in animals and plants. In these, abiotic factors such as lack of water or high temperature can influence their emission. ROS play an important role in physio- and pathological processes. Mitochondria are particularly susceptible to oxidative damage, as electrons escaping from the ETC electron transport chain in the inner membrane react with oxygen to produce a superoxide anion. This anion is unstable and cannot cross membranes, but it is rapidly converted to hydrogen peroxide, which is permeable to the membrane. It can then undergo the Fenton reaction to produce the hydroxyl radical, which is highly reactive in the mitochondrial matrix. Elevated levels of ROS lead to increased mitochondrial DNA (mtDNA) damage.•O2− and H2O2. However, they also come from sources such as peroxisomes, xanthine oxidase (XO), lipo- and cyclo-oxygenase and cytochrome P450 in endoplasmic reticulum [•O2− and H2O2 participate in redox signalling, but their production is significantly enhanced during oxidative stress conditions, producing an imbalance between ROS production and the action of the antioxidant defence system. They include SOD enzymes that reduce O2 into H2O2 . Transmembrane NADPH oxidases (NOXs)  and the eticulum . In mitod Mn-SOD  or SOD2 During episodes of environmental stress, oxidative damage (OS) in cells can increase dramatically, causing damage to their structures . GeneralNuclear and mtDNA have a separate evolutionary origin, as it is derived from the genomes of bacteria that were engulfed by the early ancestors of today’s eukaryotic cells. In extant organisms, most proteins present in mitochondria  are encoded by nuclear DNA. mtDNA is particularly susceptible to reactive oxygen species generated by the respiratory chain , due to DNA damage refers to physico-chemical changes in DNA , which cOxidation of DNA can lead to alterations in DNA bases or double helix breaks, among other mutagenic alterations . Hydroxy•OH radical easily leads to hydrogen abstraction , forming different products and ribose fragments  and their neutralisation by enzymes such as superoxide dismutase, catalase, glutathione peroxidase and others. Some mutations that increase ROS production ultimately induce oxidative damage to the mitochondrial matrix, inhibiting proteins such as Sirtuin3 (antioxidant and anti-tumour protein)  and reduAging mitochondria are the critical factor in the origin of neurodegenerative diseases. In the brains of individuals with Alzheimer’s disease, there is high oxidative damage to nDNA and mtDNA, approximately 10-fold greater than in people without the disease. In Huntington’s disease, the mutant huntingtin protein causes mitochondrial dysfunction, with higher levels of ROS and increased oxidative stress .During ageing, changes in mtDNA are well documented in the literature. Such changes include an accumulation of oxidised base pairs, base mismatches, strand breaks and deletions. Specific mutations and haplogroups of mtDNA emerge as predictors of both lifespan and risk of various age-associated diseases: cancer, diabetes, heart failure, sarcopenia and Alzheimer’s and Parkinson’s disease . In neur+ delivered to the intermembrane space is returned to the matrix during the oxidative phosphorylation reaction, so there is a reducing environment within the latter, facilitating the conversion of ADP into ATP , pass through the chain and reduce O2 in the mitochondrial matrix prematurely and incompletely to superoxide anion  species .Mitochondrial genome mutations accumulate in somatic tissues during normal ageing, as well as increased ROS production and biomarkers of ROS damage . DemonstCell membranes are sensitive to radical damage due to the presence of polyunsaturated fatty acids. Another main effect of ROS is lipid peroxidation, which occurs when membrane phospholipids are brought into contact with an ROS oxidising agent. In this reaction, the free radical oxidises an unsaturated lipid chain, leading to the formation of a hydroperoxidised lipid and an alkyl radical. This lipoperoxidation results in alterations of the membrane structure, affecting its fluidity and damaging its integrity . This prThe association of oxygen-derived free radicals with polyunsaturated fatty acids results in a number of extremely reactive electrophilic aldehydes during the process of lipid peroxidation. This effect happens as a result of continuing free radical chain reactions before they are terminated. Studies establishing a correlation between this type of oxidative disruption, neurodegeneration, and disease offered a wealth of information .Peroxidation, which leads to atherosclerosis, is believed to occur inside blood vessel walls rather than (or to a lesser extent) in LDL circulating in the blood. Just as LDL can enter vessel walls, minimally modified LDL (LDL with some oxidation but not enough to be recognized by scavenger receptors) can escape back into the circulation. As a result, the sensitivity of circulating LDL to peroxidation can be a potentially valuable biomarker, suggestive of peroxidation in blood vessels .The initiation phase of lipid peroxidation . If a frLipid peroxidation can give rise to toxic breakdown products such as hydroxynonenal. Once formed, the peroxy radical cycles to a four-linked cyclic peroxide. The process increases with the number of unsaturations of the polyunsaturated fatty acid. Thus, when free radicals attack arachidonic acid (with four unsaturations) the initial abstraction of a hydrogen atom can occur at three points in the carbon chain of the fatty acid, because it has three methylene groups (-CH2-) attached to a carbon atom flanked by double bonds, thus increasing the complexity of the peroxidation reaction. The fatty acid undergoes a further reaction with oxygen followed by a break in the cycle to give hydroperoxynonenal, which is reduced to hydroxynonenal . PatientPeroxidation of arachidonic acid leads to cyclic peroxides such as isoprostanoids in addition to hydroperoxides . AlternaIn lipid peroxidation, toxic aldehydes such as malonaldehyde A–C and hProteins and enzymes are large, complex molecules that perform critical functions in the body. They are encoded by nuclear and mitochondrial DNA and do most of the work in cells. They are necessary for the structure, function and regulation of the body’s tissues and organs. Another critical aspect of OS is the damage triggered to the structural integrity of proteins, causing loss of catalytic activity of several enzymes and paralysis in the regulation of metabolic pathways. In the last 20 years, research linking lipid peroxidation and neurodegeneration has increased dramatically, particularly with the advent of proteomics, as each disease has become better understood. Not only in neurodegenerative diseases, but also in tumours, this recent area of research has given critical knowledge about protein modifications.Unlike nucleic acids, oxidised proteins must be hydrolysed or processed by the proteasome to prevent their diffusion in the metabolic network or their interaction with other proteins. The effects of ROS on proteins are several: (i) oxidation of amino acid residues, (ii) cleavage of peptide bonds and (iii) aggregation between proteins. A wide range of diseases have been linked to the presence of oxidised proteins, such as Alzheimer’s disease, rheumatoid arthritis and others .The nature of free radical-mediated protein damage depends on the amino acid composition .2O2 scavengers [− can produce several harmful by-products, and oxidation of ribose can induce strand breaks in bacterial DNA [ROS can pass through bacterial membranes and damage their nucleic acids, proteins and cell membranes. Consequently, bacterial pathogens employ various strategies to avoid the negative consequences of ROS production, such as directly preventing their secretion by using secreted effector proteins or toxins that interfere with the translocation of the NADPH oxidase complex, or even interfering with the signalling pathways required for their activation . Severalavengers . Free iravengers . DNA damrial DNA .2O2 in a process called chemotaxis [Neutrophils are normally found in the bloodstream (where they live for about a day), and in humans they constitute 40–70% of all white blood cells. They are formed from stem cells in the bone marrow and differentiate into neutrophil-killer and neutrophil-agent subpopulations, forming an essential part of the innate immune system. Neutrophils are short-lived and highly mobile, as they can be found in tissues where other cells are not. During the initial phase of a bacterial infection or in some cancers, neutrophils are one of the first cells to migrate to the site of inflammation, through blood vessels and then interstitial tissue, following chemical signals such as interleukin-8 (IL-8) and peroxide Hemotaxis .Neutrophils are highly mobile and rapidly congregate at the focus of infection, attracted by cytokines expressed by activated endothelium, mast cells and macrophages. They recruit and activate other cells of the immune system, playing a key role in the first line of defence against invading pathogens, with three methods of attacking micro-organisms: phagocytosis (ingestion), degranulation  and generation of neutrophil extracellular traps (NETs).NADPH oxidase is an enzyme complex found in the cell membrane, in the outer space. It is found in the membranes of phagosomes used by neutrophilic white blood cells to engulf micro-organisms. This enzyme catalyses the production of a superoxide anion by transferring an electron to oxygen from NADPH .ROS generated by NADPH oxidase play an important role in host antimicrobial defence and inflammation, activating animal immune responses and plant signalling. Superoxide anion directly kills bacteria and fungi, as the virulence of many pathogens is attenuated when their superoxide dismutase (SOD) genes are knocked out. In essence, insufficient activity can lead to increased susceptibility to microorganisms, but excessive action can lead to oxidative stress and cell damage. Careful regulation of NADPH oxidase activity is crucial for maintaining a healthy level of ROS in the body .Vascular NADPH oxidases are regulated by a number of hormones and factors involved in vascular remodelling and disease, such as thrombin, platelet-derived growth factor (PDGF), tumour necrosis factor (TNF-α), lactosylceramide, IL-1 and oxidised LDL, by agonists and by arachidonic acid . MutatioThe formation of lipid hydroperoxides by neutrophil-derived ROS can also cause DNA damage. This process will produce a number of highly reactive side products, such as epoxides and aldehydes. Malondialdehyde and 4-hydroxynonenal, for example, have been widely researched and shown to be extremely DNA reactive and mutagenic .Moreover, neutrophils play crucial roles in liver repair by promoting the phenotypic conversion of proinflammatory Ly6ChiCX3CR1lo monocytes/macrophages to proresolving Ly6CloCX3CR1hi macrophages. ROS expressed by neutrophils are important mediators that trigger this phenotypic conversion to promote liver repair. This conversion is prevented by neutrophil depletion through anti-Ly6G antibody, genetic deficiency of granulocyte colony-stimulating factor or genetic deficiency of NADPH oxidase 2 (Nox2) . AdoptivAntimicrobial peptides (AMPs) are mainly gene-encoded peptides, generally positively charged, with molecular weights less than 10 kDa. The majority of AMPs have small amino acid residue spans, ranging from 5 to 40, displaying diverse amino acid sequences and secondary structures forming amphipathic helixes. AMPs can bind to the microbial cell wall or membrane through electrostatic and hydrophobic interactions, disrupting the membrane . AMPs arFurthermore, unlike traditional antibiotics, which target a certain metabolic enzyme and can cause microbe resistance, AMPs destroy microorganisms primarily through a process requiring membrane destruction, which is normally difficult for microbes to overcome resistance . The ami2+-dependent NADH oxidation that dramatically increased oxidized NADH levels, suggesting that elevated oxidation was induced by mitochondrial Ca2+ [2+ levels inhibited NADH oxidation, which is consistent with the assumption that NADH oxidation necessitates the entrance of Ca2+ ions into the mitochondria. These findings were consistent with PMAP-23 inducing a Ca2+-dependent NADH oxidation. Increased levels of ROS disrupt intracellular redox homeostasis decreasing GSH levels and changes the intracellular environment to a more oxidative state. PMAP-23 induced apoptosis in C. albicans cells via mitochondrial Ca2+-induced ROS. The overproduction of mitochondrial ROS compromised C. albicans’ intracellular redox homeostasis and decreased glutathione levels in fungal cells, resulting in oxidative stress [Recently, the antimicrobial activity of antimicrobial peptides has been related to the production of ROS. For example, PMAP-23, a member of the cathelicidin family in pigs, induces a Caial Ca2+ . Reducede stress .Antimicrobial strategies such as ROS may be a feasible choice for coping with the increasing antimicrobial resistance situation. ROS have the potential to be a powerful weapon in the fight against microbial infections since they are toxic to a wide variety of pathogens by inducing oxidative stress . ROS-indInside cell and mitochondria, the harmful effects of ROS include structural changes in DNA and RNA, peroxidation of membrane phospholipids (composed of polyunsaturated fatty acids) and oxidation of cellular proteins that perform numerous functions inside the cell. It is therefore important to keep ROS levels at physiological values, preventing them from increasing and triggering the expression of the mitochondrial protein UCP2 on its inner membrane . The unc+ protons from the mitochondrial intermembrane space to its matrix, thus contributing to the reduction of molecular O2 to H2O. The disruption of this process is known as the Warburg effect [UCPs are located in the same mitochondrial inner membrane as ATP synthase, which is also a proton channel. Both proteins work by introducing protons from the intermembrane space into the matrix, UCP2 generating heat and the other synthesizing adenosine triphosphate ATP from adenosine diphosphate ADP and inorganic phosphate, the last step of oxidative phosphorylation OSPHOS . OXPHOS g effect .Saccharomyces cerevisiae prefers glucose fermentation to ethanol over aerobic respiration in the presence of a high concentration of the former [Around 1920, Otto Heinrich Warburg and his group deduced that glucose and oxygen deprivation in tumour cells leads to lack of energy and ultimate apoptosis of the cells. Biochemist Herbert Grace Crabtree extended Warburg’s research by discovering environmental or genetic influences. Crabtree observed that e former . Warburge former . This Wae former . Cancer e former . Thus, te former .2 to H2O. Activation of the UCP2 protein occurs by an increase in ROS emitted [•O2−, Mitochondrial respiration is coupled to ATP synthesis via this ADP phosphorylation step, i.e., protons introduced by ATP synthase are used to reduce molecular O emitted , thus prIn neoplastic cells, mitochondrial metabolism is modified to meet increased energy needs and the requirements associated with rapid and uncontrolled proliferation. One of the most prominent changes occurs in cellular energy metabolism, and is known as the.An additional benefit of the Warburg effect is the detour of ETC substrates to decrease mitochondrial ROS production . Cancer Zhang et al., 2006, demonstrate that, consistent with in vitro studies, overexpression of UCP2 leads to tumour development in vivo in an orthotopic model of breast cancer. Genipin, a phytochemical molecule, suppresses the tumorigenic properties of UCP2, mediated by downregulation of reactive oxygen species and downregulation of UCP2. This study demonstrates that: (i) the Warburg effect is mediated by UCP2; (ii) this protein is overexpressed in breast cancer and many other cancers; (iii) it promotes tumorigenic properties in vitro and in vivo; and (iv) genipin suppresses the tumour-promoting function of UCP2 .The endoplasmic reticulum (ER) is an organelle present in most eukaryotic cells, specialised in protein folding and transport. Alterations in the ER environment leads to the accumulation of misfolded proteins and this seriously affects various cell signalling processes, such as reduction-oxidation (redox) homeostasis, energy production, inflammation, differentiation and apoptosis. Reactive oxygen species (ROS) have been linked to cellular stress, and play a key role in many cellular processes occurring in the cytosol and in various organelles, such as the ER and mitochondria. Alteration of ROS homeostasis in the ER is sufficient to cause ER stress. It is also unclear how changes in the protein folding environment in the ER causes oxidative stress. Furthermore, it is unknown how ROS production and protein misfolding induce cell apoptosis and contribute to several degenerative diseases .The unfolded protein response (UPR) is a group of adaptive signalling pathways whose function is to resolve protein misfolding and restore an efficient environment for protein folding. UPR signalling arises in response to protein misfolding in the ER. Prolonged ER stress leads to activation of the pro-apoptotic UPR, which plays a critical role in physiological and pathological conditions .Haemolytic anaemias are caused by haemolysis, i.e., the abnormal breakdown of red blood cells (RBCs), either in the blood vessels (intravascular) or elsewhere in the human body (extravascular). During its course, red blood cells are destroyed faster than they can be made. Oxidative stress aggravates the symptoms of many diseases, including haemolytic anaemias, as it has been found in sickle cell anaemia and thalassaemia, glucose-6-phosphate dehydrogenase deficiency, hereditary spherocytosis, congenital dyserythropoietic anaemias and paroxysmal nocturnal haemoglobinuria. Although oxidative stress is not the origin of these diseases, OS of erythroid cells plays a crucial role in haemolysis due to ineffective erythropoiesis in the bone marrow and poor survival of red blood cells in the circulation. In addition, platelets and polymorphonuclear white blood cells (PMN) are also exposed to OS .Correction of anaemia by red blood cell transfusions or iron supplementation may increase the oxidative stress burden. This situation suggests that both iron and redox status should be monitored during treatment, using red blood cells as biomarkers .Oxidative stress is an early event in the aetiology of several diseases, as biomarkers of oxidative stress appear early in their development. The high chemical reactivity of ROS makes them very effective weapons against most biomolecules. Oxidants are implicated in an increasing number of varied and dynamic processes in molecular and cell biology research, where the underlying chemical pathways are often unknown. We here aimed to link chemistry and biology, and to put what could be considered a solid mechanistic foundation. We hope this review can help students and researchers to understand the mechanism of these highly reactive molecules and their role as mediators of oxidative modifications of cellular components.


Prescription opioids are used for some chronic pain conditions. However, generally, long-term therapy has unwanted side effects which may trigger addiction, overdose, and eventually cause deaths. Opioid addiction and chronic pain conditions have both been associated with evidence of genetic and epigenetic alterations. Despite intense research interest, many questions about the contribution of epigenetic changes to this typology of addiction vulnerability and development remain unanswered. The aim of this review was to summarize the epigenetic modifications detected in specific tissues or brain areas and associated with opioid prescription and misuse in patients who have initiated prescribed opioid management for chronic non-cancer pain. The review considers the effects of opioid exposure on the epigenome in central and peripheral tissues in animal models and human subjects and highlights the mechanisms in which opioid epigenetics may be involved. This will improve our current understanding, provide the basis for targeted, personalized pain management, and thus balance opioid risks and benefits in managing chronic pain. Chronic pain represents significant public health concerns and prescription opioids are a common treatment option for cancer pain management, for end-of-life treatment, in relation to surgery, and for short-term use in severe acute/chronic pain conditions not related to cancer . The nonOpioid drugs act not only in nociceptive processes but also in modulating gastrointestinal, endocrine, and autonomic functions, as well as in affecting cognition and reward systems . The relμ-opioid receptor 1 (OPRM1), the cytochrome P450 2D6 (CYP2D6), the catechol-O-methyl transferase (COMT) genes and in the ATP-binding cassette family genes have been found to be associated with differences in morphine consumption and metabolization process [Nevertheless, it is important to note that the lack of consistent findings regarding the identification of personal risk factors that may predict opioid misuse in chronic pain patients was recently evidenced in the literature . Among t process . The mai process ,13.An intrinsic mechanism that can be induced and modulated by environmental stimuli and that represents the bridging gap between environment and genetics is epigenetics . SpecifiThe aim of this review was to report the collected data from published studies on the identified epigenetic modifications associated with opioid prescription and misuse in patients who have initiated prescribed opioids for pain.The literature search strategy included the use of PubMed, MEDLINE, Google Scholar, and Embase databases. Publications up to February 2021 were included. Peer-reviewed articles were collected based on the search terms: prescription opioids, pain, epigenetics, gene expression, DNA methylation, non-coding RNAs, chromatin, and histone modifications. Relevant literature was selected based on headline and abstract. A comprehensive search was conducted in order to provide an outline of the detected epigenetic changes related to the administration or assumption of prescription opioids in the context of pain. We also used broad search terms to increase the probability of precise identification of the target articles .All the publications collected in the literature search phase underwent abstract screening to determine eligibility for full-text data extraction. To be eligible for data extraction, studies met the following inclusion criteria: (1) the selected publications were in English language only; (2) the samples were composed of animal models to study chronic pain or human subjects with chronic noncancer pain (persistent pain lasting longer than 3 months), (3) participants were exposed to or were using prescription opioids, (4) the abstract listed one or more of the following terms in reference to potential epigenetic changes: DNA methylation, histone, chromatin, non-coding RNAs, microRNAs, gene or protein expression. Studies related to genetic polymorphisms, which are changes in the DNA sequence, were excluded from the review.Finally, to identify potentially relevant studies not detected through the main screening, a few articles were selected from other reviews.A thorough screening of the collected papers was conducted based on the inclusion criteria and quality of the experiments . Analyzing the identified papers, three main categories were evidenced and grouped in Moreover, within the three subgroups, the articles were listed in the tables by the type of opioids . It thus became clear that the identified studies primarily investigated the morphine exposure effect on gene expression and chromatin modifications. Among the studies exploring oxycodone exposure in animal models, experiments measuring DNA methylation and DNA hydroxymethylation were selected. The few studies that considered these effects on human subjects were focused on DNA methylation. Interestingly, the screening revealed a group of articles related to the intergenerational and transgenerational effects of the epigenetic marks identified. Hence, the related remarkable information was described in a dedicated paragraph. In light of the few human studies identified, a paragraph about the epigenetic research related to heroin users was included to represent how these modifications have been studied in human subjects and to give future directions for the prescription opioid research related to different pain conditions.locus ceruleus, rostral ventral medulla, and in the substantia gelatinosa of the dorsal horn. The major mechanism through which opioids relieve pain is the stimulation of descending inhibitory neurons through activation of the μ opioid MOP receptors. Common prescription opioids responsible for this opioid-induced analgesia effect are morphine, codeine, tramadol, fentanyl, hydromorphone, buprenorphine, hydrocodone, oxycodone, oxymorphone, methadone, and tapentadol [Opioid receptors are widely distributed both centrally and in the periphery, particularly in the periaqueductal grey, pentadol .Opioid medications are often prescribed for acute episodes of pain for short-term use or for cancer-related pain. Opioids are also used for chronic non-cancer pain in selected cases when non-opioid and adjuvant therapies have failed and other pain medications have proven ineffective. These drugs are defined as highly effective and safe analgesics when used appropriately and included in a multifaceted strategy by competent clinicians .However, since opioid-induced pain relief and addiction do not act through distinct mechanisms in distinct brain areas, opioid drugs could not only have a simple analgesic effect but also affect or compromise the ability to feel pleasure and socialize as well as the reward system . Neural In light of the fact that the opioid analgesic efficacy often decreases with continuous use and that patients with refractory complex chronic pain are at high risk of abuse, a two-level strategy might be set up to contrast the opioid crisis. The first level would include prescription monitoring programs and dose limitations to prevent abuse/misuse; the second could increase research at the molecular level to identify the central and peripheral mechanisms underlying the drug action and to explore precision medicine options.The term epigenetics refers to the study of heritable changes in the gene function that do not involve changes in the DNA sequence . EpigeneDNA methylation, the addition of a methyl group on the 5th carbon of the DNA cytosine, is shown particularly relevant in CpG islands, regions of the genome containing a large number of CpG dinucleotide repeats . It reguThe chromatin modifications include those related to histone tails, such as histone methylation, chromatin remodeling, and post-translational modifications affecting electrostatic nucleosome interactions. These changes have been clinically and pre-clinically evidenced to be associated with opioid exposures . ConcernThe third important regulator of transcriptional activity is represented by ncRNAs , i.e., RThe nociceptive response was demonstrated to activate these epigenetic mechanisms by modulating pain genes and possibly mediating the transition from acute to chronic pain. Studies highlight that also opioids are involved in diverse types of epigenetic regulation and thus they might influence the analgesic effects or the increased risk of continued opioid intake and development of a substance use disorder following long-term opioid therapy ,58.However, the identification of specific factors associated with the individual opioid response or with side effect vulnerability has just been launched. The molecular mechanisms through which some individuals develop negative consequences associated with prolonged prescription opioid use, including hyperalgesia, addiction, sleep problems, hypogonadism, fractures, and surgical failures ,60 are pOPRM1 gene. The OPRM1 is responsible for a significant portion of the variance in the response to morphine. Pharmacologically relevant doses of morphine were chronically administered. Microarray gene expression profiling of brain areas, relevant for nociception, analgesia, conditioned reinforcement, and drug abuse , revealed specific genes  and gene networks associated with analgesic tolerance and predisposition. Instead, the dopamine D3 and D4 receptors, the dopamine transporter, DARPP-32, and calcyon genes were found to be differentially regulated by morphine, and it has been hypothesized that they are associated with morphine-rewarding effects. All these genes modified by chronic morphine administration play a role in the neuroadaptation pathways [nucleus accumbens (NAc) was also reported at both the global level and in specific loci. In particular, reduced H3K9me2 was revealed in the FosB gene with subsequently enhanced activity of the encoded protein ΔFosB, a key player in drug behavioral response, and in the glutamatergic genes, such as GRIN2A, GRM5, GRM8, with corresponding increases in their mRNA expression. The study also demonstrated that downregulation of G9a after chronic morphine enhanced locomotor sensitization and sensitivity to the rewarding effect of the drug. At the same time, G9a downregulation reduced the physical dependence and delayed the morphine analgesic tolerance development, highlighting its crucial role in maintaining normal gene transcription [The studies exploring the effect of painkillers in animals mainly investigated gene and protein expression changes following morphine exposure. One of the first studies determined the analgesic tolerance to morphine in mice with congenic genotypes for a proximal region of chromosome 10 that includes the pathways . The effcription .BDNF) expression through the repression of G9a and a subsequent reduction of the repressive mark H3K9me2. Further, knockdown of G9a evidenced an increased sensitivity to pain and to morphine reward-related behavior [Other mechanisms were also proposed to be related to morphine reward and tolerance. Concerning morphine-seeking behavior and reward, a mechanism that may contribute to increased response in the behavioral preference for opioids under pain conditions was proposed. In fact, persistent pain and repeated morphine upregulated MeCP2, a transcriptional regulator which binds to methylated CpG sites of DNA, in the mouse central nucleus of the amygdala (CeA) . This stbehavior .GRIA1 promoters (the GluA1 gene) with the subsequent de-repression of GRIA1 expression and enhancement of the morphine-seeking behavior in rats with pain experience [Subsequent experiments of morphine self-administration in rats with persistent inflammatory pain demonstrated that after morphine withdrawal GluA1 subunits of glutamate AMPA receptors, known to be upregulated in pain conditions with the potentiated nociceptive transmission, were overexpressed in CeA. The proposed mechanism by which pain determines GluA1 upregulation was the decreased recruitment of MeCP2 onto perience .OPRM1 gene in the dorsal root ganglia (DRG); this resulted in condensed chromatin and gene silencing and a diminished opioid analgesic effect. Consistently, G9a inhibition in the injured DRG rescued the MOR expression and restored the morphine effect on pain hypersensitivity induced by nerve injury [C/EBPβ (CCAAT/enhancer-binding protein β), a transcription factor regulating gene expression in immune and inflammatory responses [C/EBPβ was shown to indirectly inhibit MOR and the voltage-gated K+ channel Kv1.2 abundance in the ipsilateral DRG by activating the G9a gene (EHMT2), thus resulting in condensed chromatin and gene silencing. To confirm that C/EBPβ contributes to neuropathic pain through epigenetic silencing of Kv1.2 and MOR, it was demonstrated that blocking C/EBPβ the morphine analgesia following CCI was rescued [Concerning analgesia, some studies reported epigenetic changes associated with neuropathic pain and morphine analgesia. First, it was demonstrated that the diminished expression of the μ-opioid receptors (MOR), observed following nerve injury, was mediated by an epigenetic mechanism. In fact, nerve injury consistently increased the G9a action and thus the levels of H3K9me2 in the promoter of the e injury . Li and e injury . Specifiesponses . C/EBPβ  rescued .BDNF, FosB gene expression and with CREB (cAMP-responsive element-binding protein) activation; all factors implicated in drug addiction and learning memory [Another fundamental aspect explored was the long-term neural and behavioral consequences after opioid exposures in the context of histone tail modifications. The repressive histone tail mark H3K27me3 was identified specifically in adolescent mice VTA following oxycodone exposure with consistent inhibition of gene regulation indicating an age-dependent oxycodone effect . Insteadg memory .BDNF has also been identified among DNA methylation-regulated genes. Barrow and coworkers (2017) observed that acute morphine exposure induced significantly decreased methylation in BDNF and IL1B, and significantly increased methylation in IL6 and NR3C1. While chronic morphine exposure induced significantly decreased methylation of BDNF and COMT and significantly increased methylation of IL1B and NR3C1 in different rat brain regions. The study also analyzed global DNA 5-hydroxymethylation observing a significant increase in the cerebral cortex, hippocampus, and hypothalamus, as well as a significant decrease in the midbrain [Moreover, the midbrain .BDNF transcription and translation. One study demonstrated that this process, which contributes to the development of chronic tolerance to morphine analgesia in mouse DRG, was mediated by miR-219 by targeting CaMKIIγ [It is also important to report the role that micro-RNAs (miRNAs) might play in opioid response and pain. In fact, it has been observed that commonly used analgesics altered their expression . One exa CaMKIIγ . Another CaMKIIγ .SYN and PSD95 genes with consistent higher expression of these synaptic proteins and the synaptic density [SYNAPSIN, SHANK2, and GAP4, were observed, too [Other studies focused on the rewarding effects of the widely abused prescription drug oxycodone, exploring related gene expression and DNA methylation changes after a period of chronic oxycodone self-administration. In particular, RNA-sequencing in the NAc and caudate-putamen (CPu) of mice following extended 14-day oxycodone self-administration revealed differential expression of the axon guidance molecule integrins, semaphorins, and ephrins, which may correspond to alterations in the axon-target connections and synaptogenesis and thus to the oxycodone-induced neuroadaptations . In addi density . In the ved, too . In addived, too ,32.The reported epigenetic changes in connection with the related brain areas are reported in Research on epigenetic effects of prescription opioids in humans is still in its infancy and mainly focuses on DNA methylation changes in peripheral tissues. The reason for the scarce results may be seen in light of the difficulties to measure epigenetic modifications in the human brain: the majority of the differentially methylated patterns was detected in peripheral tissues that may not be indicative of alterations in reward and pain pathways. However, other studies identified overlapping epigenetic signatures in peripheral and central tissues indicating that blood cells could represent a source of non-invasive biomarkers for opioid misuse susceptibility or prediction also in chronic pain patients ,65. The PARG (the poly ADP-Ribose glycohydrolase gene), which is highly expressed in the human brain; cg04983519 at the EHMT2 gene encoding for the histone methyltransferase G9A involved in transcriptional repression; cg21381136 at the RERE gene (arginine-glutamic acid dipeptide repeats gene) that was shown to interact with EHMT2; and cg18177613 related to CFAP77 (the Cilia And Flagella Associated Protein 77 gene) [OSBPL9, oxysterol binding protein-like 9) and the protein binding/axon guidance .Three differentially methylated CpG sites in genes implicated in chromatin remodeling, DNA binding, cell survival, and cell projection were identified with decreased DNA methylation associated with opioid dependence development. Total DNA derived from whole blood samples was compared between opioid-exposed subjects developing dependence and not developing dependence. In particular, the top CpG sites identified were: cg17426237 related to 77 gene) . Other COPRM1 promoter. At a genome-wide level, the DNA methylation changed in 1701 CpGs belonging to genes related to natural killer cell-mediated cytotoxicity and cellular defense response; in particular, the methylation decreased within a week (2.7 ± 1.5 days) of the last opioid dose and re-increased 39 ± 10 days after the last opioid dose in the majority of these CpGs (>65%), while the other CpGs had an opposite trend [Interestingly, one study explored the early epigenetic effect of therapeutic opioids at three time points; one before and two after opioid self-administration in 33 subjects undergoing standard dental surgery and never exposed to opioids. The study demonstrated how the response to opioids occurred within the first few days to weeks following drug exposure as dose-dependent hypermethylation was observed in nine CpGs analyzed in the te trend .TLR4) and the μ-opioid receptor (OPRM1). However, the global level of DNA methylation was shown to be predictive for persistent pain, comparing 70 women with persistent pain diagnosis with 70 matched controls who had not developed persistent postsurgical pain [Even if related to cancer pain, the experiment of Kringel and coworkers (2019) should also be considered because of the prospective design, which may provide more definitive answers to questions of causality. The study confirmed the predictive utility of epigenetic testing in a sample of 140 women who had undergone breast cancer surgery . DNA metcal pain .Evidence suggests that epigenetic changes might be transferred to the next generations. Furthermore, germ cells express opioid receptors strengthening the potential of this research. Experiments related to the reactivity to opioids suggest that alterations in the endogenous opioid system can persist in the progeny of parents exposed to opioids and in the following generations . Most ofOPRM1 promoter was revealed. This change was found to be associated with a worse outcome of neonatal abstinence syndrome [in utero; interestingly, the detected miR-504 increase correlated with increased expression of DRD1 and DRD2 receptors in synapses. [Both intergenerational and transgenerational epigenetic inheritance refers to the transmission of non-DNA base sequence information between generations; but only in the second case, the effects are found in generations that were not exposed to the initial signal or environment that triggered the change ,68. Intesyndrome . A more syndrome , and theynapses. . Gilardiynapses. ,40. One ynapses. . AnotherThe studies investigating intergenerational and transgenerational epigenetic changes following prescription opioid exposure are reported in SNAP25, synapsin 2a, and 2b, pallidin, amysin, and the endogenous opioid peptide prodynorphin genes, were found significantly reduced in heroin abusers [GABRD) gene were found in the NAc following heroin self-administration. The study proposed new therapeutic strategies based on manipulation of the enzymes responsible for this epigenetic modification, such as DNMT1 and DNMT3A [FYN gene, its kinase activity, and the phosphorylation of its target. Interestingly, inhibition of FYN activity attenuates heroin self-administration in rats [Even if the research on how epigenetic modifications could contribute to addiction development has been less comprehensive for heroin compared with psychostimulants , importa abusers . Moreove abusers . Also ind DNMT3A . Pharmac in rats .BRG1) with related higher expression of this gene in the mPFC after heroin self-administration [Novel therapeutic targets were also proposed among histone tail modifications ,76. For stration . The actstration .The role of miRNAs, 18-25 nucleotide non-coding sequences with a wide range of regulatory roles in gene expression and neuronal functions, was also investigated in heroin-seeking behavior. MiR-218 was found to regulate many neuroplasticity-related genes and target the methyl CpG-binding protein 2 (Mecp2), thus inhibiting heroin-induced reinforcement .EGR1 and EGR2 expression and increased the methylation of the EGR2 gene promoter. EGR1 and EGR2 are two early response genes sensitive to heavy substance use and the environment [Heroin studies also recall the role of environmental factors affecting the vulnerability to the drugs of abuse response and may also modulate the neuro-anatomical/neuro-chemical impacts of uncontrolled drug use and relapse propensity. Environmental enrichment, such as the presence of alternative rewards that reduce stress and anxiety, was demonstrated to significantly reduce motivational measures of drug-seeking and drug-taking behavior, also in heroin self-administration models. From a molecular point of view, it was demonstrated that both forms of reward, heroin or environmental enrichment, reduced the ironment .Even if the heroin epigenetics research seems to be at an advanced stage compared with the epigenetic research of prescription opioids, it should be noted that many questions remain unanswered. The length of time to observe the epigenetic changes induced by chronic heroin, especially in the peripheral tissues that are more suitable for biomarker identification, still needs further evaluation .The present overview elucidates the detected epigenetic modifications following prescription opioids for pain. It is worth noting that the majority of studies focused on morphine administration in animal models. Few studies analyzed epigenetic changes in humans after different opioid administration, however, the evidenced brain areas involved in prescription opioids action and their role in pain perception are reported in G9a expression with subsequent gene expression activation of ΔFosB and glutamatergic genes [One of the most interesting results is the role of the histone methyltransferase G9a, which specifically methylates the Lysine 9 of histone H3 (H3K9me1 and H3K9me2). G9a seems to act as a crucial player balancing gene expression and resulted in turn differentially regulated by morphine exposure or pain. In fact, repeated morphine administrations downregulate the ic genes . Interesic genes . GluA1 gic genes . Consistic genes .G9a expression and subsequent OPRM1 gene silencing in DRG [By contrast, nerve injury models and CCI resulted in enhanced g in DRG ,23 and tThe role of G9a was also evidenced in peripheral samples of opioid-dependent subjects in which a decreased DNA methylation was evidenced in a specific CpG of the gene. Even if DNA methylation is mainly associated with gene repression, it is difficult to predict the consequence of a single CpG methylation on the gene and protein expression and this result should draw further attention to the role of G9a.This evidence corroborates the hypothesis that epigenetically mediated G9a regulation is a common adaptive response of neurons to compensate for the negative effects of chronic drug use on the brain , and thiBDNF expression with associated higher global 5hmC and the process was shown to involve specific miRNAs. Moreover, BDNF is a well-known mediator of the mechanism through which neuronal plasticity is affected by environmental experiences [Another key element detected from the literature analyzed is BDNF and its miRNA-mediated regulation. Chronic morphine administration induced an increased eriences  and shoueriences ,85. Inteeriences . In thiseriences  are accoeriences ,89. Thiseriences .These findings highlight the complex interplay between pain, the adaptive responses of the immune system, addiction, and the environment, offering a possibility to reduce or prevent the negative consequences of chronic pain and prescription opioid abuse by environmental and lifestyle factors.It is well known that the environment  can influence the epigenome and gene expression. In contrast, it is still a controversial and debated issue whether the environment experienced during our lifetime may be inherited intergenerationally or transgenerationally and thus if it can have an impact on the health of the descendants. However, new insights are emerging ,92,93,94Although few, the human studies reveal crucial aspects to be considered for future research design. In fact, the cited experiments involved the comparison of subgroups of opioid exposed subjects, who developed or not developed the diseases (persistent pain or opioid dependence). In other cases, the epigenetic analyses were conducted at different time points, before and after opioids assumption. These strategies allow to prove the strength of an association between a disease and potential causal factors: prospective longitudinal investigations are encouraged in prescription opioids and pain research to identify epigenetic biomarkers.The studies on heroin point toward the possibility of developing new epigenetic pharmacological approaches. In particular, research should fully explore the role of DNMT, HDAC, and other potential therapeutic targets identified in the context of heroin rewarding and heroin-seeking behavior in order to determine if these targets might be considered in the research of prescription opioid use disorders. It should be noted that the findings of the studies oriented towards identifying promising therapeutic targets for heroin use disorders mainly evidenced the GABAergic and glutamatergic transmission . ImportaOur review summarizes the epigenetic factors that are associated with prescription opioids used for pain. Specific biomarkers that could serve as potential therapeutic targets or factors allowing to reveal the complex molecular mechanisms underlying prescription opioid addiction and pain are reported. Of special interest is the histone methyltransferase G9a, the role of which should be explored in detail. The studies conducted in humans are insufficient for the purpose of biomarker identification and the underlying epigenetics remains poorly understood. From these studies, the first evidence of the causal relationship between therapeutic opioid administration and epigenetic changes was revealed and research should further explore the temporal dynamics of these modifications in response to both prescription opioids administration and pain.Exploring these pathways could reveal the molecular mechanisms and potential therapeutic targets for preventing chronic pain and addiction.