4.1 Receptor Signaling
The first gene group that showed differences between control and MMT groups belonged to Receptor Signaling via the opioid receptors (the binding site for morphine and its components). Preclinical experiments increase KOR and MOR mRNA expression in NAs after morphine exposure. 41Also, preclinical and clinical experiments report that chronic cocaine and amphetamine treatments increased MOR expression. Upregulation of MOR with morphine treatment was detected in Human and Monkey immune cells. 42 Moreover, upregulation of MOR has been reported in methadone maintenance patients. 43 KOR upregulation has been reported in the immune and brain region of rats under chronic morphine treatment. 44,45 Our results show upregulation of MOR and KOR in MMT, which agrees with these previous reports. This is not surprising since the elevation of MOR and KOR expression in addiction has been reported in previous preclinical and clinical studies. 46,47 However, there are contradicting results of Kappa, Mu, and delta receptor downregulation in PBMC during methadone maintenance on heroin-addicted subjects. 48 Coordinately, MMT increased the expression level of DOR in this study. To our knowledge, there is little information for Delta OR mRNA alteration in morphine components induction. Although, the morphine-induced upregulation of DOR cell surface via selective agonist for MOR has been confirmed. 49, another study showed no effect of prolonged morphine on DOR protein level and suggested that the increased DOR density on the cell membrane is due to intracellular receptor reserve. 50 In an animal experiment, no significant difference was detected in DOR mRNA levels between morphine treatment and controls; therefore, no receptor neosynthesis mechanism was observed during morphine induction. 51 We assumed that methadone as a MOR agonist could upregulate the MOR and KOR. On the other hand, DOR upregulation could be mediated via MOR in chronic drug exposure based on previous reports. since heterodimerization of mu and delta-opioid receptors is involved in morphine tolerance 52 and previously in vivo functional study has been reported the co-expression of these receptors in brain areas located in circuits related to drug reward 53 likewise, reduced stress induction in the presence of Delta opioid agonists have been observed during withdrawal in rodents. 12MOR and DOR agonists inhibited GABA interneurons in the hippocampus resulting in increased pyramidal cell activity, facilitating learning and memory processing through drug use. 31 These findings parallel our results that the opioidergic pathway is activated after long-term methadone abuse, mimicking the OUD process. We also detected upregulation of KOR expression in our drug-abuse cohort. Conversely, another report demonstrated that the expression level of the KOR mRNA in PBLs was significantly decreased in MMT and drug abuse groups 43, which contrasts with our results. We suggest that the elevation of KOR expression may be related to methadone acting as an unspecific agonist for the KOR receptor. Therefore, KOR antagonists may function as pharmacotherapeutic agents by reducing drug craving and consumption. We assumed that one of the compensative mechanisms to overcome receptor desensitization is to extend the ligand's contact area and enhance the sensitivity to the lowest amount of ligand. In turn this mechanism develop tolerance and leads to displaces of the stimulation threshold, which demands more and more ligands. However, we could not differentiate that this Simultaneously increasing of opioid receptors related to methadone effect as an agonist or retained from chronic drug abuse must be assessed in future study. Moreover, further study is needed to clarify this finding at the protein level.
4.2 Signal Transduction
The excitation of dopamine in the nucleus accumbens could be through µ-opioid receptors on VTA-DA neurons. 54 The activation of the VTA-DA neurons of the limbic system provides a better understanding of the brain circuitry involved in reward, motivation, addiction, and neuropsychiatric illness.
The DRD1 gene expression level increased significantly under methadone treatment in the present study. According to several reports, increasing D1 dopamine receptor expression and activity in key brain regions is essential for reward and may contribute to drug dependency. 55-57 Glutamate release in the pyramidal cells of the basolateral amygdala (BLA) has mediated by the presynaptic D1 receptor overexpression, suggesting that synaptic transmission may switch from inhibition to excitation in chronic morphine treatment. 58 therefore, our results are in agreement with these findings.
Several addiction studies have reported DRD2 reduction. 59-61 However, there is a contradiction since upregulation of the DRD2 gene in opioid abusers has also been observed. 62 However, they assessed the expression level of DRD2 protein in VTA of postmortem brain of OUD patients and assumed that DRD2 elevation during chronic opiate abuse could increase the reward. 62 Our results disagree with their finding, and one reason could be that they investigated the VTA region. We assume that an increased level of DRD2 could contribute to the acute phase of drug abuse. During the chronic phase and adaptation development, expression alteration will occur. Several efforts have been made to use DA agonists as treatment approaches in addiction (in vivo & in vitro). The results suggest reduced self-administration of the drug by overexpression of DA receptors. 59,63-65 Moreover, peripheral D2 receptor antagonists would escalate impulsive behavior. 66 Compulsive behavior has been observed in mice with D2 receptor knockdown in the striatum, representing an addiction-like phenotype. 67 However, overexpression of the D2 receptor appears to attenuate alcohol consumption and cocaine self- administration. 68,69 Furthermore, similar to dopamine transmission, drug administration demonstrates reduced D2 receptor expression level in the animal model (high or low impulsivity). 70 In the present study, we demonstrate the significant increase in DRD2 gene expression level under methadone treatment. In opiate abuse, MOR regulates DA release in the dopaminergic synapses. Afterward, DA binds to the Dopamine receptors and DRD2 as well. DRD2, an inhibitory GPCR, activates the punishment pathway by inhibiting the indirect ventral striatal signaling through decreased intracellular cAMP. 71 Functional study on OUD patients showed that reduced DRD2 expression correlated with relapse and drug-seeking behavior. 71,72 Despite DRD1, DRD2 has a high affinity for DA binding. Therefore, a low level of DA could have occupied the DRD2, in addiction during firing, DA release following drug abuse, the excitatory pathway through DRD1 induces. On the other hand, reduction in DRD2 expression mediates dysregulation of motivation and in accompanied by craving behavior. As previously reported, postsynaptic overexpression of DRD2 in NAc in mice showed increased motivation. 73 Due to incapability for experience rewarding in low availability of DRD2 in striatal, the high threshold is demanded. 72 This preliminary evidence represents tolerance development. Ultimately increased DRD2 expression under methadone maintenance may consider as the positive effect of methadone as the first cure line of addiction (FDA approved agonist) in modulating the DA receptors expression.
Bromocriptine, a DRD2 agonist, was investigated for drug abuse treatment. Bromocriptine is an agonist (DRD2) and a partial antagonist (DRD3). 63 functional study in morphine-dependent mice showed that bromocriptine could function as morphine analgesia and inhibit tolerance development; however it promotes opiate withdrawal signs. 74 Although full agonists such as bromocriptine reduce drug self-administration and relieve drug withdrawal symptoms, utility is limited by high abuse liability. 75 Therefore, continued investigation regarding any efficient DRD2 agonist with a reduced side effect in the long term is needed for future study.
Dopamine D3 receptors are located mainly in the nucleus accumbens, VTA, and amygdala, representing dependence paths in the brain. Moreover, pharmacological, human post mortem, and genetic studies have been supported the role of DRD3 in drug dependency. 76 In the present study, the mRNA expression level of DRD3 was downregulated in the MMT group. One probable strategy could be utilizing DRD3 ligands to reduce relapse in the abstinence stage since the limited direct involvement DRD3 in drug reinforcement. 76 however, Preclinical data indicate an up-regulation of DRD3 expression following exposure to DA-elevating drugs, including cocaine, nicotine, alcohol, methamphetamine (MA) polydrug users, and methadone maintained subjects. 77 Preclinical studies demonstrate the beneficial effect of DRD3 antagonists in decreasing motivation to self-administer drugs and diminishing craving. 77 in agreement with previous functional evidence, we assumed that downregulation of DRD3 in the MMT group highlights the effects of methadone in decreasing relapse.
Dopamine D4 receptor is expressed in the striatum, cerebral cortex (CTX), and hippocampus where neuronal functions are not well deﬁned. 78 A signiﬁcant reduction has been reported in dopamine D4 receptor mRNA expression in abstinent, as well as in heroin and alcohol. However, in OUD undergoing MMT, DRD4 reduction was insignificant, the same as our study group. Thus, it seems that the reduction of dopamine signaling through the dopamine D4 receptor is a risk factor for drug dependency. 78 Functional study by DRD4 blockade demonstrated attenuated craving and nicotine-seeking behavior. Therefore, DRD4 antagonists deduce to be effective therapeutic agents to reduce relapse in tobacco addiction. 79,80 Considering these findings, we suggest a potential role for DRD4 as a novel strategy for treating drug dependence. Hypermethylation of DRD4 has been observed in heroin abused, which resulted in the DRD4 receptor expression level alteration in the addicted subjects. These results are considered evidence for DRD4 role in drug addiction pathophysiology. 81 Dopaminergic deficiency involves in the pathophysiology of depression. A significant reduction of DRD4 mRNA in PBMC belonged to the patients with major depression was observed. 82 Recently, a meta-analysis study in Iran and around the world was performed and showed the positive effect of methadone on depression in OUD. 83 Our data show no signiﬁcant difference in the peripheral blood lymphocytes of MMT and control group. According to our results and a similar previous study. 78 We assume that methadone could positively impact the DRD4 receptor by restoring the expression level and attenuating the significant differences with the control group. So, as a result, one of the benefits of methadone treatment could be modulating depression in OUD, and DRD4 antagonists could be considered the target of future studies. However, this suspicion is also raised that this result may be because this receptor's naturally low expression level in PBMCs impeded detection or simply that methadone did not impact the DRD4.
The data demonstrated a reduction of DRD5 mRNA expression level in MMT compared to the control group, which supports the pivotal rule of DRD5 in behavioral patterns connected to OUD. Overall, this data supports increasing responsiveness to drugs and hypersensitivity, congruent to dependence and tolerance strategies. There are some discrepancies between the literature regarding DRD5 expression in OUD. Significant downregulation of the DRD5 has been reported in computer game addicts. Also, increased DRD5 and signaling through this receptor decrease responsiveness to cocaine. 84 However, increased DRD5 gene expression and activation pathways (G-proteins) have already been described as modulators of OUD. 85,86 In another report, the DRD5 expression level in OUD subjects and the MMT cohort was not statistically signiﬁcant. 78 This discrepancy seems to be attributed to brain location-dependent DRD5 expression, as DRD5 expressed significantly in the prefrontal cortex and NAc compared to the hippocampus in OUD patients. 87
The COMT enzyme regulates dopamine degradation in the brain. It has a pivotal rule in DA turnover, and we assumed that any changes in the expression level of this enzyme would impact the rate of DA degradation. Furthermore, the reduction of COMT could elevate DA levels in extracellular areas. Therefore, COMT reduction is a rational process after methadone treatment, which could be a compensating mechanism in DA depletion conditions to avoid withdrawal symptoms.
4.3 Resistance related to Intracellular Transport
DNM1L encodes a large GTPase dynamin 1 like protein, which has an essential role in regulating postsynaptic clathrin-mediated endocytosis and mitochondrial fission/fusion. significant up-regulation of DNM1L gene expression in the CTX of animal models for alcohol dependence and microglia exposed to cocaine have been reported previously. 88,89 However, our research group reported that DNM1L gene expression was not significantly affected by treatment with methadone and DAMGO in addiction. 27 Downregulation of miR-331-5p, which targets DNM1L, has been reported in ketamine abusers. 90 Also, upregulation of DNM1L led to altering mitophagy in T and B cells and Auto-antibody production in Systemic Lupus Erythematosus. 91 Upregulation of DNM1L was associated with rheumatoid arthritis (RA). DNM1L upregulation by induced ROS productio causes oxidative stress. DNM1L inhibitor was used for RA. 92 DNM1L overexpression associated with proliferation, invasion and, apoptosis in gastric adenocarcinoma. 93
In the present study, we demonstrate that the mRNA expressions level of the DNM1L gene is decreased in MMT patients. DNM1L plays a role in synaptic vesicle recycling and is pivotal in receptor internalization pathways. Reduction of DNM1L could disrupt synaptic transmission through mitochondrial fission/fusion imbalanced and caused neural activity impairment. 94 We suggest that this reduction of DNM1L could disrupt the DA or opioid receptor desensitization due to long-term drug abuse and impairment in synaptic transmission. As previously reported that mitochondria depletion could lead to axonal mitochondria depletion, axonal degeneration, and neuronal death. 94
Rab proteins are Ras-related GTPases that have a pivotal role in regulating different endocytic steps and maintaining several cellular homeostatic pathways. 26 A study on rapid induction of tolerance by opiate suggested that morphine promotes MOR endocytosis which can cause desensitization and ultimately induces morphine tolerance and dependence. RAB22A is one of the downstream components in this endocytosis network. 95 Recently, we reported DNM1L gene expression was not significantly affected, but RAB22A gene expression decreased with methadone treatment. Thus, the DNM1L gene could be involved in cellular pathways of morphine-induced tolerance, and it could explain the difference between morphine and other similar Mu opioid receptor agonists. 27 RAB22A gene overexpression has been reported in different human cancer types such as Breast, Pancreatic, Lung, Osteosarcoma, which have a significant part in autophagy, invasive behavior, migration, and migration tumor development through vesicular trafficking and exosome formation. 96-99 recently, many research types have focused on RAB22A inhibition using miRNAs to prevent tumor cells from progression and metastasis. However, to our knowledge for the first time, the present study assessed the RAB22A expression in OUD patients. The present study demonstrates that the RAB22A1 gene expression in MMT patients was not significant. During the treatment period, methadone may moderate RAB22A in the MMT group, which could normalize the activity of RAB22A. 100 Alternatively, RAB22A mRNA level could compensate for DNM1L expression reduction to overcome tolerance development.
4.4 Protein–Protein Interaction (PPI)
We assess three protein groups involved in different steps, including receptor signaling, signal transduction, and resistance. Generally, in system biology, proteins functions in a complex, not separately. In the same cellular process, all the participating gene products interact. The PPIs study could elucidate the function of each protein within the cell. One of the advantages of PPIs study is that the undefined function of the protein could be predictable according to the other related proteins' function. 101 The accurate exploration of PPIs facilitates the identification of functional pathways to accelerate the molecular mechanisms underlying cellular processes. Therefore, with the help of PPI, we assess the relation between these three groups and highlight the other link protein and pathways that are potentially involved in OUD pathophysiology.
To illustrate any connection between addiction-associated genes, this investigation constructed the PPI networks and provided clustering and enrichment analysis. MAPK signaling pathway demonstrates the most significant enriched pathway within the highest scored cluster during ontology analysis of the intersection MCODE clusters. The enriched GO terms corresponding to cellular components show myelin sheath as an impacted area (GO:0043209)(Fig 4). The speed and efficiency of neuronal communication depend on myelination's degree. 102 However, there are areas of demyelination in different brain regions in alcohol and tobacco users103,104. Decreased myelination could contribute to impulsivity behavior in OUD remains unclear and require further research and testing. Behavior (GO:0007610) and learning or memory (GO:0007611) are highlighted for biological processes. MAPK-ERK signaling pathway plays a critical role in generating long-term stable alterations underlying learning and memory in addiction. 105 As shown in (Fig 5), most hub nodes demonstrate targets that have a pivotal role in the MAPK-ERK signaling pathway.
Methadone is used as a usual medication for pain relief and drug addiction. However, long-term MMT has some disadvantages as well. A better understanding of dependence and tolerance during MMT is revealed by linkages between the most critical hubs and their involved pathways extracted from the theoretical network analysis and the experimental section results and clarify the advantages and disadvantages of methadone as a therapeutic strategy. In long-term MMT, methadone (a full agonist of opioid receptors) binds to the opioid receptors, particularly the µ-opioid receptor (MOR). And stimulates intracellular downstream signaling, including adenylate cyclase-cAMP/protein kinase A. In the acute phase of opioid abuse, MOR decreases gamma-aminobutyric (GABA) release in VTA and triggers DA release in NAc. 106 Instead, in chronic opioid use, increasing GABA release in the synaptic cleft of VTA occurs. Afterward binding of GABA to GABA receptor in postsynaptic neuron action potentially inhibits dopamine cell firing in NAc. During chronic MMT, upregulation of opioid receptors intensifies the GABA release inhibition, over-activates PKA in MOR downstream signaling, and improves adaptation. On the other hand, we observed reduced DNM1L expression which, causes decline and blunting of receptor desensitization process and may develop tolerance. In response to reducing DA release in NAc dopaminergic synapse, we hypothesize that increased receptor expression of D1 and D2 consider as a compensation mechanism. Also, COMT reduction is another mechanism to preserve DA level in the presynaptic neuron in NA.
The upregulation of DRD2 in MMT patients may represent a therapeutic prospect. DRD2 through GNAI1 could impact RA1GAP, which activates RAP1A and RAP1B by exchanging GDP with GTP. The activation of RAP1A follows this, and RAP1B functions on downstream paths, including PI3K-AKT signaling pathways and MAPK signaling pathways. Previously an in vivo study on cocaine administrated mice reported that a novel DA-PKA-Rap1-MAPK intracellular signaling mechanism is activated by DRD1 could have regulated reward-related behavior. Rap1 stimulates MAPK (ERK) signaling and mediate neuronal excitability. 107 Another in vivo study showed that over-activation of DRD2 impacts the A2aR-PKA-Rap1-MEK pathway and mediates aversive behavior. 108 On the other hand, D1R activation by methadone could shift from PKA to ERK intracellular signaling cascades.
ERK functioned as a crucial downstream kinase in the second messenger signaling pathway to mediate drug addiction, such as morphine. Elevation of ERK phosphorylation by addictive drugs has been reported previously. 109,110 which triggers the rewarding behavior. 111 Therefore, activation of ERK seems to be critical for drug cravings 112, and high persistent activity of ERK observed after a long period of withdrawal. Furthermore, in vivo studies demonstrated the involvement of ERK signaling in the reward drug-induced in the midbrain (MB). 113
Increased ERK phosphorylation has been reported in drug reinforcement. 114 Moreover, the functional study reported the memory disruption by utilizing ERK inhibitors, which decreased the ERK1/2 phosphorylation level. 115 These findings highlight the pivotal role of ERK in memory preserving.
While the ERK dephosphorylation exact mechanism is not adequately understood, several studies have focused on the ERK activation mechanism. 116 Targeting upstream signaling pathways mediated by ERK dephosphorylation could be a therapeutic approach that needs further investigation. Consequently, the dual‐specificity phosphatase (DUSP) superfamily (ERK dephosphorylation enzymes) indicated benefits in diminishing addiction memory. 116The amygdala microinfusion of ERK1/2 inhibitor was previously utilized to decrease anxiety-related behavior in naive rats. 117 Also, the Y3214996, a potent, selective, ATP-competitive ERK inhibitor, was used in xenograft models (harboring ERK pathway alteration) to overcome resistance. This ERK inhibitor improved cancer treatment efficacy and demonstrated antitumor activity, recommended for future phase I clinical trials. 118 Also, another ERK inhibitor, ulixertinib on the clinical trial, was reported to be more efficient and well-tolerated in different tumor types and introduced for use in combination therapy for MAPK-driven cancers. 119 However, there is a lack of any investigation for ERK inhibitor in OUD treatment strategy.
The list of most critical and related hubs in OUD pathogenesis pathways, which could be introduced as new potential therapeutic targets, is provided in the present study. Our study supports the therapeutic impacts of methadone on OUD through mimicking opioid signaling and upregulation of DRD2. Previously some evidence reports the involvement of ERK1/2 in the pathogenesis of synaptic plasticity and memory in addiction. 120,121 Also, a functional study on rats reported the elevation of ERK activity in NAc shell during morphine administration, and also ERK inhibitors prevent conditioned place preference. 122 ERK inhibitors or biomarkers (hubs) in the MAPK signaling pathway could be potential targets for further investigation.
The present study has some limitations. This study focuses on mRNA expression analysis; however, some mechanisms such as mRNA degradation and mRNA stability that could impact protein level are not excluded. Although, the cautious and considerate use of PBMCs as a surrogate for brain gene expression may additionally be warranted. Alternatively, further functional study with large sample size or comparative studies between tissue and blood on mammalian organisms would be informative. While future studies using a larger sample size are needed to validate our results. This case-control observational study provides essential information regarding genetic and receptor expression differences that may underlie the behavioral differences in OUD.
We propose that the D2 receptor has therapeutic potential for OUD, and using D2 agonist directly could replace methadone therapy to bypass opioidergic signaling. Also, ERK activation is a downstream pathway influenced during D2 upregulation. The inhibition of this pathway may also have a positive therapeutic impact in OUD. Further, studies investigating these two pathways can provide an efficient strategy for combating addiction while avoiding the side effects caused by the current therapeutic options.